Podocin participates in the assembly of tight junctions between foot processes in nephrotic podocytes

The predominant type of cellular junction between normal podocyte foot processes is the slit diaphragm. Under nephrotic conditions, however, foot process effacement leads to the loss of slit diaphragms and the new formation of tight junctions composed of the proteins coxsackievirus and adenovirus receptor (CAR) and zonula occludens 1 (ZO-1). Podocin, a protein that plays a key role in maintaining the integrity of the slit diaphragm, has also been localized to these tight junctions, but its function at this site is unknown. In this study, we confirmed that podocin colocalizes with CAR and ZO-1 at the tight junction between foot processes in nephrotic rats. Using primary cultures of rat podocytes, as well as cell lines that co-expressed podocin and CAR, we observed that podocin was recruited to sites of cell-cell contact and that it co-localized with CAR and ZO-1. Immunoprecipitation suggested that these three junctional proteins from a multi-protein complex. Consistent with this, we found that podociin facilitated the coalescence of preassembled lipid rafts containing CAR and restricted their lateral mobility, the latter likely a result of dynamic actin reorganization and subsequent tethering of CAR-podocin complexes to the cytoskeleton. In conclusion, in addition to serving as a structural protein of the slit diaphragm of normal podocytes, our data suggest that podocin may also serve as a scaffold that links tight junction proteins to the actin cytoskeleton in nephrotic foot processes.